Cholesterol regulates the rate of sterol synthesis by controlling the level of microsomal 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase, the first enzyme specific for cholesterol synthesis. The long-term goal of the proposed investigations is to isolate and quantitate the macromolecules involved in controlling the level of HMG-CoA reductase. The potential regulatory sites at which exogenous cholesterol could act to suppress the synthesis of HMG-CoA reductase are: a. A decrease in the level of HMG-CoA reductase specific messenger RNA. b. Less efficient translation of reductase mRNA due to a decrease in ribosome initiation frequency or a lower rate of translation or release. c. An alteration in membrane or protein structure which prevents integration of newly synthesized HMG-CoA reductase into the endoplasmic reticulum and results in accumulation of an inactive cytoplasmic pool of enzyme. Reductase levels in the endoplasmic reticulum and in any soluble pool which may exist will be quantitated using antibody against the purified protein and a previously developed anti-antibody technique. The rate of reductase synthesis and degradation will be determined in cell cultures of animal and human origin. Binding of iodinated antibody will be used to quantitate the number and size of HMG-CoA reductase synthesizing polyribosomes and investigate potential translational control. The level of translatable HMG-CoA reductase specific mRNA will be measured in a cell-free protein synthesizing system derived from wheat germ. Individuals with defective cholesterol regulation of HMG-CoA reductase and cholesterol synthesis exhibit serum hypercholesterolemia and atheroschlerosis. These individuals are the source of established cell culture lines which will be used to help elucidate the site and mechanism of cholesterol regulation of HMG-CoA reductase synthesis. Cholesterol regulation of reductase synthesis represents a useful model system for molecular investigations of the mechanisms controlling gene expression and protein synthesis in animal cells.